Fluorouracil (FUra) is widely used in the treatement of a number of cancers. The active metabolite of this drug is thought to be FdUMP, which is known to inhibit thymidylate synthetase and thus abolish de novo synthesis of dTMP. However, FUra is also known to be incorporated into RNA and to affect RNA maturation and function. Both of the aforementioned may represent possible modes of FUra cytotoxicity, and these may vary in different cells. Attempts have been made to correlate cytotoxic effects of FUra with the activities of enzymes involved in FdUMP formation, with drug incorporation into RNA, and with FdUMP or dUMP pool sizes, but the primary mode of cytotoxicity of this drug remains unclear. We have developed methods which allow us to simultaneously monitor and quantitate the following aspects of intracellular FUra metabolism: (1) the covalent complex formed between FdUMP, CH2-H4 folate, and thymidylate synthetase, (2) drug incorporation into RNA, and (3) FUra nucleotides in tissue culture cells. With these methods, we shall be able to assess FUra metabolism in several cell lines and hopefully ascertain rational biochemical reasons for the differential responses of these cells to FUra. We may also be able to correlate one or more of the biochemical parameters measured with cell death, and thus establish the primary mechanism of cytotoxocity of this drug. The extension of these studies to human colon and breast tumor cells will be the first step in the extension of these methods to screening tumor biopsies; this latter step will hopefully aid in predicting the responsiveness of tumors prior to the initiation of chemotherapy.